JP2009538854A - Isolated natural natural collagen - Google Patents

Abstract

  The present invention relates to isolated collagen, a method for producing and isolating collagen from collagen-containing tissue, and the use of the isolated collagen in a biomatrix as an in vitro test system, tissue substitute or organ substitute About. This collagen is extracted with urea.

Description

  The present invention relates to isolated collagen, a method for producing and isolating collagen from collagen-containing tissues, and the isolated collagen in a biomatrix, in vitro-test system, tissue substitute or organ substitute. Related to using as.

Background Art Extracellular matrix (ECM) or carrier structures for cell culture, so-called biomatrix, are usually produced from isolated collagen, ie from matrix proteins isolated from collagen-containing tissues. Such biomatrix plays an important role, especially in regenerative medicine. In this case, so-called tissue engineering has the purpose of preserving or replacing diseased, missing or lost tissue- or organ function. For this purpose, tissue- or organ structures are usually cultured in vitro, in which at least one tissue-specific cell type is inoculated on a pre-made carrier structure or biomatrix and subsequently cultured. The biomatrix and the cells cultured thereon form a tissue-or organ equivalent or substitute.

  In order to realize a tissue-specific function, it is necessary for cells applied on the biomatrix to regenerate or newly form a matrix protein. In order to successfully cultivate cells on such a carrier structure, it is important that a matrix protein having a native or natural identity characteristic exists as much as possible.

  Known biomatrixes are usually produced from freshly prepared collagen-containing tissue, eg extracted from skin or tendon, ie from isolated collagen.

  A known extraction method is extraction using acetic acid water. In conventional extraction protocols, acetic acid is used at a concentration of 0.1% to 500 mmol / l. In order to extract collagen from collagen-containing tissues such as rat tail tendons (RTT), the tissues are agitated with acetic acid for 20 hours to 7 days, usually at room temperature. It is a drawback that the final concentration of collagen in the extraction fraction is limited based on the high viscosity of this collagen solution. In particular, the original structure of the extracted matrix protein is attacked during the incubation based on acid hydrolysis with acetic acid and enzymatic digestion with proteolytic enzymes normally present, in which case the matrix protein is partially or completely denatured. It is a drawback. Therefore, the subsequent regeneration of the original native collagen structure and the reconstitution of the biological function of the matrix protein are limited. When the matrix protein isolated and partially denatured by acetic acid extraction is subsequently lyophilized, the properties of the collagen product reconstituted using the lyophilizate are significantly biased from the original collagen, Its use for the culture of tissue specific cells is severely limited. Furthermore, biomatrix produced from matrix proteins isolated using acetic acid extraction exhibits further disadvantages, such as uncontrollable (usually one-dimensional longitudinal) shrinkage.

  The use of biomatrix produced by such known methods for in vitro test systems, for example for the testing of new agents, is significantly limited. This is because the cultured cells develop physiological and pathological properties and cellular functions that are often adversely altered compared to in vivo or natural location-states due to the presence of denatured matrix proteins. Because it does. Therefore, the transferability of in-vitro test results becomes more difficult.

  There is a need for selective and improved means and methods for easily obtaining improved, i.e., sufficiently natural, isolated collagen and biomatrix produced therefrom without the disadvantages of the state of the art. .

The technical problem underlying the present invention is mainly to provide a method and means for producing, obtaining or isolating collagen or matrix protein from collagen-containing tissue. In this case, isolated collagen has a high proportion of matrix proteins having a structure that is naturally equivalent. Another technical challenge associated with this is to provide an improved biomatrix that is suitable for the production of organ- or tissue substitutes or equivalents or in vitro test systems. Another technical challenge linked to this is the in vitro test system, the state of the art for analysis and diagnosis of infection and for the study and testing of denatured or genetically altered human or animal cells. It is an object of the present invention to provide a diagnostic agent and a therapeutic agent that overcome the above-mentioned drawbacks.

  This underlying technical problem is essentially solved by a method for isolating collagen or collagen matrix proteins from collagen-containing tissue according to the features of claim 1.

  According to the present invention, collagen-containing fibers are isolated from the collagen-containing tissue in step (a), and in the subsequent step (b), the isolated collagen-containing fibers are treated with 5-15 mol / l of urea, in particular Incubation in an aqueous urea solution containing a concentration of 7-12 mol / l, particularly preferably about 9 mol / l, releases or elutes the collagen-containing fraction from the fibers during or by this. In the subsequent step (c) according to the present invention, when the dissolved collagen-containing fraction is separated from the fiber and tissue residue, a collagen-containing aqueous solution is obtained. Advantageously, in another step (d), urea is separated from the collagen-containing fraction, and advantageously in another step (e), the urea-free collagen-containing fraction is restored, preferably in a buffer. When recovered, reconstituted and isolated collagen is obtained in an aqueous solution.

  Accordingly, the present invention also contemplates incubating the isolated collagen-containing fibers with a substantially high concentration of urea solution for a period of time, thereby eluting the collagen matrix protein from the tissue. Surprisingly, the extraction according to the invention makes it possible to isolate a matrix protein which is essentially equivalent to nature and can be almost completely reconstituted in aqueous buffer after separation of urea. The circular dichroism-spectroscopy (DC) of the collagen fraction isolated according to the present invention proves the typical so-called “triple helix” structure of natural collagen, which is specifically 217 in DC-spectrophotographs. It is characterized by a negative absorption band of ˜227 nm and possibly a weak negative absorption band around 200 nm. The UV-spectral examination (UV) of the collagen fraction isolated according to the present invention also shows the characteristic amino acid signature of histidine at 213 nm, which also shows the intact native protein structure. UV is therefore also very suitable for the simple qualification of collagen matrix proteins, without the need to use known antibody tests that often show adverse cross-reactions. Therefore, another subject of the present invention is also a method for isolating collagen or collagen matrix protein, where the concentration of collagen is measured by UV.

  Advantageously, the collagen isolated according to the invention is used to obtain a biomatrix and from this a tissue- or organ substitute or equivalent, or in particular an in vitro test system which no longer exhibits the known disadvantages of one-dimensional contraction. Can do. The growth of tissue-specific cells and the new synthesis of the matrix by the cells from the biomatrix takes place to the same extent as the biomatrix improved or at least obtained by known methods.

  The collagen-containing tissue (from which collagen-containing fibers are isolated in step (a)) used in one preferred embodiment of this method is the rat's tail tendon isolated from the rat's tail. In one variant, the collagen-containing tissue is advantageously an azellarisierter porcine small intestine.

  In step (b), the urea solution is preferably stirred together with the collagen-containing fibers. In step (b), the collagen-containing fibers are incubated with the urea solution for 12-36 hours, particularly preferably about 24 hours, advantageously under agitation, to elution collagen-containing fractions from the fibers particularly effectively. Is advantageous.

  In step (c), it is advantageous to separate the dissolved collagen-containing fraction from the fiber and tissue residues in a manner known per se by mechanical separation, preferably by centrifugation. In another preferred variant, the separation is carried out selectively or additionally by filtration.

  For the particular field of use, it is appropriate in step (c) to separate and in some cases completely separate the collagen-containing fractions dissolved by fractionation using gel filtration. Subsequently, in some cases, a plurality of separated collagen fractions can be combined. One skilled in the art can thus select a suitable and / or tissue specific matrix protein composition for each field of use.

  The isolated collagen-containing fraction or the collagen fraction separated by fractionation is advantageously subjected to analytical methods. The characterization of the matrix protein is preferably performed by DC. In another preferred variant, this characterization is performed selectively or additionally by UV. In another preferred variant, this characterization is performed selectively or additionally by ESI-MS / MS. In another preferred variant, this characterization is performed selectively or additionally by MALDI-TOF / TOF. Protein fractions can be separated and isolated by one-dimensional or two-dimensional gel electrophoresis, isoelectric focusing and / or SDS-PAGE by methods known per se.

  In order to separate the urea contained in the solution from the collagen-containing fraction, it is advantageous to carry out a gradient dialysis in step (d). In this case, it is advantageous to dialyze in the cold, ie preferably at a temperature of 0 to about 10 ° C., preferably about 4 ° C., for a period of 4 to 12 days, advantageously 7 days. Dialysis against water is advantageous. The aqueous collagen solution thus produced preferably has a collagen concentration of about 3 to about 8 mg / l.

  In one variant of the invention, the dialyzed fraction is advantageously concentrated 2 or 3 times to a specific collagen content. This resulting concentrate is particularly suitable for further separation on SDS-PAGE, for further characterization and for the production of a boil-stable biomatrix.

  The collagen fraction is then preferably reconstituted in a further step (e) in a phosphate buffered saline solution (PBS) or similar medium or buffer system. This restoration allows the matrix protein to be used for the production of an improved biomatrix since it essentially re-deploys all natural properties and original structural parameters.

  The object of the present invention is also an isolated collagen that can be produced by the method of the present invention or is produced by the method. The aqueous collagen solution obtained according to the invention has a high proportion of undenatured native collagen in the aqueous medium, in particular the total collagen in the solution ≧ 50%, in particular ≧ 60%, ≧ 70%, ≧ 80 %, ≧ 90%, particularly ≧ 99%. Such collagen has a triple helix-signature characteristic triple helix-signature, especially in circular dichroism-spectroscopy (see above).

  In another preferred embodiment of the present invention, the resulting collagen-containing fraction aqueous solution is lyophilized to provide a storable dry collagen product. Subsequent addition of water or a suitable medium to the lyophilized collagen thus obtained advantageously results in almost completely the original three-dimensional structure of the collagen. There is no destruction of the natural triple helix-structure by lyophilization. Another object of the invention is also lyophilized collagen obtained according to the invention. Therefore, when the aqueous collagen fraction is freeze-dried by a method known per se, a storable dry collagen product is obtained. It is of course also possible to store the solution in the intermediate state in the frozen state, for example at -10 ° C to -80 ° C, in particular at about -20 ° C.

  Another subject of the present invention is a method for producing a biomatrix, which first carries out steps (a) to (e) of the method of the present invention, thereby producing an aqueous collagen solution or a collagen-containing fraction. In another step (f), the resulting collagen solution and cell culture medium or the like are mixed in a ratio of 2: 1 to 1: 2, preferably 1: 1, to obtain a collagen-containing matrix precursor solution. Cause it to occur. The collagen content of this solution is advantageously 3-8 mg of collagen per ml of solution, preferably 5-7 mg of collagen per ml of solution. In another step (g), the matrix precursor solution thus obtained is gelled at an advantageously elevated temperature, preferably 21 ° C. to 37 ° C., into a collagen-containing biomatrix. This collagen gel can also be obtained by dissolving the freeze-dried collagen product obtained according to the present invention.

  Therefore, another object of the present invention is a biomatrix that can be produced by the above-described method or that is preferably produced using the same. Another object of the invention is also a biomatrix containing or preferably consisting of collagen isolated using the method of the invention. The present invention particularly relates to a gel-like biomatrix for use in culture methods, for example, for culturing cells of a specific tissue type or cells of multiple tissue types. The combination of biomatrix contemplated by the present invention and cells cultured therein can be used for the production of in vitro tissue- or organ test systems.

  “Biomatrix” is understood to be a gel structure containing collagen, cell culture medium, buffer and optionally serum. Preferred cell culture media are DMEM (Dulbecco's Modified Eagle Medium) and M199. A preferred buffer system is Hepes-buffer. As serum, fetal bovine serum (FCS) or human, especially autologes Serum, is advantageously used. In a preferred embodiment, the pH value of the solution from the cell culture medium, buffer and serum is 7.5 to 8.5, for example 7.6 to 8.2, in particular 7.8. Of course, depending on the field of use, the biomatrix can contain further elements such as growth factors, proteoglycans, glucosam licans, adhesives, antibiotics, selective agents and other extracellular matrix components.

For the production of a cell-containing biomatrix, it is advantageous to add the precultured cells to cell culture medium, serum and buffer, preferably several times (x-fold) concentrated, per ml It is advantageous to use 1-2 × 10 ⁵ cells, preferably 1.5 × 10 ⁵ cells per ml. Subsequently, it is mixed, for example in a ratio of 1: 2 to 2: 1, with the collagen solution obtained according to the invention when cold at 0 to 10 ° C., in particular at 4 ° C. The mixing ratio (volume) of the collagen solution and the cell suspension (buffer, serum, cells, culture medium) is advantageously 1: 1, where in the case of x-fold concentrated gel solutions In particular, the amount ratio of the collagen solution to the gel solution is preferably (x-1): 1. Subsequently, the suspension is pipetted into a culture vessel and gelled at an elevated temperature, in particular 37 ° C. (this usually takes several minutes) and then overlaid with culture medium (in-water culture). This biomatrix can be cultured for 2 days, for example. Subsequently, additional cells of still other tissue types can be seeded and cultured thereon.

  A preferred embodiment of the present invention includes a three-dimensional gel-like biomatrix for the growth of animal or human cells and for the production of three-dimensional animal or human in vitro tissue- or organ test systems. Animal or human cell culture is included. The results obtained using the tissue- or organ test system according to the invention can be expressed more strongly than the results obtained in animal examinations and ensure good transmission to humans. A further object of the invention comprises the biomatrix according to the invention and living tissue cells, particularly preferably tissue-specific cells cultured in and on or in the biomatrix, It is also a tissue equivalent or a substitute. The object of the invention is an organ equivalent comprising a biomatrix according to the invention and living tissue cells, in particular an organ-specific cell which is preferably cultured in and on or in the biomatrix. Or a substitute. In one particularly preferred embodiment, the present invention also includes the cultivation of human dermal fibroblasts in this biomatrix for the production of a three-dimensional human in vitro skin equivalent consisting of a dermal equivalent and an epidermal equivalent. Is done.

“Culture of cells” means the typical life of cells, eg fibroblast tissue, carried out in a particularly suitable environment, eg in vitro with the supply and removal of substance metabolism starting materials and products. It is understood that the function is maintained, and in particular includes cell proliferation. In the context of the present invention, dermal fibroblasts are naturally understood in particular as fibroblasts or precursors thereof which are present in the dermis or genetically altered. Fibroblasts are precursors of dermal fiber cells. Fibroblasts may be of animal or human origin: cells freshly isolated from tissue or cells of primary or genetically altered or transformed cell lines, such as WI-38, NIH / 3T3, MRC-5. This biomatrix comprises a collagen skeleton newly composed from the collagen solution obtained according to the invention at a concentration of 2-5 mg, preferably 3.5-4.5 mg of collagen per ml of bioblast and cells to be cultured. Have. This collagen skeleton is obtained according to the invention and is preferably obtained from a cell-free collagen solution, wherein the protein concentration of the collagen solution is preferably 5-7 mg / ml. For example, for the production of a fibroblast-containing biomatrix, the collagen solution is cooled, preferably at 4 ° C., in particular 5 times concentrated cell culture medium, buffer, preferably Hepes-buffer, serum, In particular a cell solution or suspension containing fetal bovine serum (FCS) and chondroitin- (4/6) -sulfate and in particular fibroblasts at a concentration of about 1.5 × 10 ⁵ / ml is added. And mix well. This mixture is gelled in about 2 minutes, for example by raising the temperature to room temperature or to 37 ° C. It is advantageous to add fibronectin onto the gel after this gelation. Fibronectin promotes cell binding to macromolecules such as collagen and adhesion to neighboring cells. The subsequent culturing of fibroblasts in the collagen gel is particularly advantageously carried out in a submerged culture (here the cells are covered with a culture medium). A cell culture medium is overlaid on a biomatrix containing fibroblasts and cultured in a manner known per se under standard conditions at 37 ° C. and 5% CO ₂ .

  In one advantageous embodiment of the invention, the fibroblasts cultured in the biomatrix can be eluted from the biomatrix again and possibly again into the biomatrix, where the cells are eluted. Later, it does not lose its specific substance metabolic capacity and its differentiation state. Therefore, the method of the present invention can also carry out an intermediate culture of fibroblasts in a biomatrix. Thus, the method of the present invention provides the advantage that sufficient cellular material can be utilized for the production of dermal and / or skin equivalents with low starting amounts of fibroblasts.

  Another embodiment of the present invention is that dermal fibroblasts whose function, morphology and / or differentiation state are to be examined are introduced into the three-dimensional biomatrix, cultured, and in this case Intended to be tested at and / or after. The invention therefore also relates to screening and diagnostic methods carried out in the use of dermal fibroblasts, in which the fibroblasts are cultured according to the method described above and / or subsequently, e.g. Can be examined for clinical, oncological, toxicological, physiological, morphological and / or molecular biological parameters. This biomatrix contains a skeleton that is composed of a collagen solution from human or animal collagen along with the fibroblasts to be cultured, ie a tissue-typical matrix protein. This collagen-fibroblast-gel is preferably subjected to submerged culture for 1-2 days according to the present invention.

In another advantageous embodiment of the invention, dermal fibroblasts can be subsequently cultured in a three-dimensional biomatrix as described above to subsequently obtain a dermal equivalent. In the context of the present invention, a “dermal equivalent” is understood to be a connective tissue-like layer formed from collagen and fibroblasts that is well matched to the original dermis. For this purpose, after 1 to 3 days, preferably 2 days, after said incubation of the gel, keratinocytes, skin hepatocytes or keratinocyte progenitors, particularly preferably pre-cultured, uncultured from human biopsy tissue Differentiated keratinocyte-stem cells are inoculated on this gel, overlaid with KBM-medium and subjected to submerged culture for 1-3 days. Complete differentiation of keratinocytes layer, air lift in CaCl ₂ to about 1.8 mmol / l-containing KBM- medium (without hEGF and BPE) - is achieved by culture. In the context of the present invention, “Airlift-Kultur” means that the height of the nutrient medium level is precisely matched to the height of the biomatrix, whereas the cells formed by keratinocytes or keratinocytes Layer means a culture that is above the nutrient medium level and is not covered by the nutrient medium, i.e. the culture is carried out in the air-nutrient medium limiting layer, with the supply of the culture medium from below. Is called. For this, the inserts from the 24-well microtiter plate are transferred into the wells of a 6 well microtiter plate, each having a diameter of 3.5 cm. Preferably after 12-14 days of airlift culture, an in vitro complete skin model consisting of skin-typical, dermal and epidermal equivalents is produced, which is advantageously used for the test method according to the invention. Can do.

  Thereafter, keratinocytes or keratinocyte-stem cells are inoculated on a biomatrix containing fibroblasts. “Keratinocytes” are understood to be cells of the epidermis that form keratinized squamous epithelium or genetically altered keratinocytes which may be of animal or human origin or precursors thereof. Since the formation of intact keratinized and well differentiated epidermis is highly dependent on the proportion of basal stem cells in the keratinocytes used, keratinocytes seeded on collagen gels are as undifferentiated keratinocytes from human biopsy tissues Advantageously, they are stem cells, ie cytokeratin 19- or integrin β1-positive basal stem cells. In this case, this is advantageously a pre-cultured cell, particularly preferably a keratinocyte passing through the first or second cell.

  Particularly preferred embodiments include only relatively high proportions of keratinocyte-cell populations having a relatively high proportion, for example 0.5%, 1%, 2%, 5%, 8% or 10% or undifferentiated stem cells. It is advantageous to use keratinocytes. Under special culture conditions (especially involving several days of submerged culture and subsequent biomatrix-system several days of airlift-culture) and the use of special culture media, these keratinocytes differentiate into a multi-layered epidermis Produces a layer. Furthermore, in a preferred embodiment according to the invention, other cells types and / or other cells of other tissue types are inoculated on this biomatrix, for example on immune system cells, before, during or after inoculation of keratinocytes. It is also intended to be able to. It is advantageously achieved that the dermal equivalent is not subjected to an undefined contraction process during the culture period.

  Accordingly, the present invention provides for the production, culture and use of skin typical and preferably human three-dimensional in vitro skin equivalents and their skin equivalents and components thereof consisting of dermal equivalents and epidermal equivalents. Also related to the method. Skin, also referred to as in vitro skin equivalent, is a typical complete skin model, especially in dermatology and allergology, where substances such as potential pharmaceuticals or cosmetics or agents such as pharmacological effects of light and heat In particular, it can be used as a test skin to test for irritation-, toxicity- and inflammatory effects and for their tolerability.

Another embodiment of the present invention includes intestinal fibroblasts and the present invention for producing a three-dimensional human in vitro-intestinal test system, preferably consisting of Caco2-cells or intestinal epithelial cells or other human cell lines. Culturing together with the biomatrix obtained by Intestinal fibroblasts are fibroblasts or precursor cells that naturally exist or have been genetically altered, especially in intestinal tissue of animal or human origin. For the production of the intestinal fibroblast-containing biomatrix according to the invention, the cell culture medium, buffer, in particular Hepes, preferably concentrated in collagen solution at 4 ° C., preferably in a volume ratio of 1: 1, preferably twice. A solution also referred to as a gel solution containing buffer and serum, preferably 10% serum and preferably intestinal fibroblasts, in particular 1.5 × 10 ⁵ / ml of pre-cultured intestinal fibroblasts Add and mix well. This mixture is gelled by raising the temperature to room temperature or to 37 ° C. The subsequent cultivation of the intestinal fibroblasts in the collagen gel is advantageously carried out in a liquid culture. This fibroblast-containing biomatrix is incubated at 37 ° C.

  Another subject of the invention is an in vitro test system comprising a biomatrix according to the invention and living cells cultured in and / or on the biomatrix. The present invention particularly relates to in vitro test systems that can be used for the analysis and diagnosis of infectious diseases and / or diseases attributed to the pathogenesis and / or parasitic microorganisms of the human or animal body, modified humans and In vitro test systems for the analysis and diagnosis of animal cells, in vitro test systems for the analysis or diagnosis of genetically altered human and animal cells, and anti-infection agents and drugs for tumors, especially static cell agents And in vitro test systems for inspecting and testing of animals and three-dimensional animal in vitro-organ- and tissue models of infected tissues such as intestine, liver, skin, cornea, trachea and mucosa.

  The cells to be examined can preferably be cultured and grown there in a three-dimensional gel-like connective tissue-like biomatrix produced according to the invention. This biomatrix contains the cells to be cultured in or on the scaffold from collagen, i.e. tissue typical matrix protein composed of collagen solution. Depending on the desired tissue type, additional other cell types, in particular other primordial cells, can be applied on this biomatrix. Using specific culture conditions and the use of specific culture media, the cells contained in this biomatrix and other cell types, preferably applied on this biomatrix, are differentiated to obtain a multilayer 3 It can be a dimensional animal tissue or organ test model. Such co-culture of animal in vitro tissue- or organ test systems and parasitic or pathogenic microorganisms according to the present invention offers the possibility to study both the infection process itself and the protective response of the corresponding organ cell systems. To do. For example, large amounts of infected cellular material and the pathogen itself can be obtained. This resulting material is then used, for example, by histological, biochemical, molecular biological or immunological methods, such as morphological changes in infected cells, pathogens such as specific substances due to toxins or To accurately study the release of proteins that are important for the expression of resistance or the release of specific substances by diseased cells, such as interleukins as a defense response, or transcription-and / or expression profiles (based on eg anti-infection Can be further analyzed to obtain a pathogenic agent as a target for drug development).

  One preferred embodiment of the present invention includes co-culture of a three-dimensional in vitro-tissue- or organ-test system produced according to the present invention with pathogenic or parasitic microorganisms. In the context of the present invention, “pathogenic or parasitic microorganisms” (also referred to herein as infectious agents) attack both eukaryotic and prokaryotic microorganisms, eg macro organisms, in particular human or animal organisms. However, it is understood to be a bacterium, fungus, protozoan, viroid, prion or virus that lives in or on the tissues of these organisms and may cause infection of these organisms, but not necessarily. The In the context of the present invention, the concept “Cokultivierung” is preferably performed in vitro, for example in the same environment suitable for both animal cells and microorganisms, for example substance metabolism starting materials and -product substances It also means the simultaneous maintenance of the vital functions of animal cells and microorganisms, in particular the simultaneous growth of cells and microorganisms, under the supply and discharge of water.

  In another embodiment of the present invention, the use of chemicals, in particular anti-infective agents or agents, in particular in the use of an in vitro-tissue- or organ test system produced according to the present invention, associated with a co-culture method. The effects on the infection process or the growth of pathogenic microorganisms can be studied. In the context of the present invention, the concept “Agens” includes in particular chemical, biological or physical means, for example light or heat that can exert a potential action on living cells. Is done.

  One preferred embodiment of the present invention involves the analysis of denatured cells. In the context of the present invention, the concept “denatured” includes all changes in normal cells, such as cell pluripotency, erythropoiesis, nucleation, polychromemia, disrupted nucleo-trait-relationship and Included are aneuploidies that can lead to disrupted differentiation or dedifferentiation and can lead to deregulated growth of cells, and are particularly relevant to cells of malignant tumors. In particular, an in vitro-tissue- or organ test system for obtaining a large amount of denatured cellular material from the denatured cells of the tissue or organ is constructed. This obtained substance is further examined in a conventional manner, for example histological, biochemical, molecular biological or immunological methods, in order to examine the release of specific substances and to obtain a transcriptional and expression profile. Be analyzed. In vitro tissue- or organ test systems composed of denatured cells are used to examine the effects of drugs and substances that are potentially suitable as drugs, in particular with regard to their ability to inhibit cell division.

  In one preferred embodiment of the present invention, patient-specifically modified cells are used to establish an in vitro-tissue- or organ test system for studying the therapeutic potential of a patient for a particular tumor disease. The

  In a preferred embodiment of the invention, it is intended in particular for the examination of genetically altered cells of said tissue or organ. In the context of the present invention, the concept “genetically altered cell” has been manipulated using genetic engineering methods (whether heterologous-DNA and / or RNA has been incorporated into the cell). Or all cells in which the unique DNA and / or -RNA has been modified, for example by deletions, inversions and additions. One particularly preferred embodiment is intended to test genetically altered cells, particularly for their function in vitro, in view of the genetic treatment of patient-specific diseases. In contrast, in vitro-tissue- or organ test systems are established using the use of such genetically altered cells.

  Finally, another subject of the present invention is the use of the collagen according to the invention for the production of tissue equivalents or -substitutes, for the production of organ equivalents or -substitutes or for the production of in vitro test systems. Is to use.

FIG. 3 shows the tissue of a fibroblast culture after 45 days in / on a biomatrix produced according to the invention.

EXAMPLES The present invention will be described in detail with reference to the following figures and examples, which should not be construed as limiting the invention.

  FIG. 1 shows the tissue of a fibroblast culture after 45 days in / on a biomatrix produced according to the present invention.

Example 1: Isolation of Collagen from Collagen-Containing Tissue Isolation of Collagen-Containing Fibers For the production of collagen solutions, tendons from rat tails are used as collagen-containing tissues. All operations are performed with aseptic material under aseptic conditions. After storage at −20 ° C., the rat tail is surface disinfected with 70% alcohol. The rat tail skin is removed and individual collagen fibers are drawn. If other starting tissues are used, optionally present cells can be carefully removed by mechanical, enzymatic or chemical treatment.

  Collagen fibers are collected in phosphate buffered saline (PBS) (pH 7.2), surface disinfected in 70% alcohol for about 10 minutes, and then thoroughly washed with PBS. Measure the weight of the fiber.

Extraction of matrix protein For extraction of matrix protein, the fibers are transferred into a high concentration urea solution; the final concentration is 9 mol / l. The batch is stirred at about 4 ° C. for about 24 hours. Subsequently, the undissolved collagen is removed by centrifugation (1000 Upm, 1 hour, 8 ° C.). Collagen is now present in solution rather than in fiber-, skeleton- or matrix form.

Gel-filtration Optionally, the collagen solution is subjected to gel filtration in other batches for separation using centrifugation. For this purpose, after extraction, about 50 ml of urea-containing collagen solution is added onto a 1.6 liter column (diameter: 6 cm, height: 60 cm) with a packing consistent with Superose ^™ 12 (GE Healthcare) and about 25 ml / h. Elute at a flow rate of. Collect 10 ml of 100 fractions each. Depending on the desired protein composition, the fractions are combined again and the others are discarded.

Gradient dialysis Gradient dialysis against water is performed to remove urea in the collagen solution. Dialysis is performed starting at 9 mol / l urea and taking 7 days at 4 ° C. until 0 mol / l urea in PBS. In this case, the collagen is fully restored, as the corresponding analysis shows.

Freeze-drying In another batch, the urea-free collagen fraction obtained from this dialysis is freeze-dried in a manner known per se to make it storable.

Example 2: Characterization of isolated matrix protein The structure of a collagen protein obtained according to the present invention, fractionated by gel filtration and free of urea by gradient dialysis against water was biophysically characterized.

CD-spectroscopy Circular dichroism-spectroscopy (CD) is used for examination of protein structure. The characteristic spectrum of the so-called “triple helix” is revealed. This shows a typical “signatur” characterized by a small negative absorption band of 217-227 nm and possibly a visible slight intensity negative absorption band around 200 nm. These spectra are clearly different from the CD-spectrum of collagen proteins of disordered structure (“random coil”), α-helix or β-pleated sheet structure. The triple helix structure is typical for collagen I, which is dominant in aqueous solution; the majority of the isolated collagen folds in water to the original native structure.

  In another batch, the collagen obtained according to the invention was lyophilized and subsequently dissolved again in water and subjected to CD-spectroscopy. It is clear that collagen solutions made from lyophilized collagen also have a characteristic triple helical structure. Collagen produced according to the present invention and lyophilized exhibits high water solubility, in which case the natural structure of the collagen is not irreparably destroyed. This property has not previously been achieved with natural source collagen.

UV-spectroscopy For demonstration of CD-results, the UV-spectrum of isolated collagen fractions was measured in the range of 190-320 nm. This UV-spectrum confirms the analysis of the CD-spectrum. There is an equilibrium between the different protein structures. That is, it is clear that when the protein concentration is doubled, the characteristic peak of the spectrum has been halved; this equilibrium shifts towards an alternative conformation.

  In a comparative experiment, the aqueous collagen solution obtained using conventional acetic acid extraction was investigated. Contrary to conventional acetic acid extracts, the collagen obtained according to the invention shows characteristic amino acid residues in the UV-spectrum. This includes in particular histidine at a wavelength of 213 nm. This enables a direct concentration measurement of the collagen obtained according to the invention, advantageously using objective spectroscopy and photometric methods.

Biochemical characterization In another batch, it was examined whether the collagen obtained according to the invention is glycosylated. Enzymatic degradation with O-glucanase disrupted the possibly present O-glycosylation. Subsequently, the proteins thus obtained were separated by two-dimensional gel electrophoresis and stained with Coomassie. Isoelectric focusing shows that the collagen obtained according to the present invention is localized in the pI-region (isopotential point) of 4.5-6 before deglucosylation. The theoretically expected value is 5.5. After deglucosylation, the characteristic “Schmier” showing the resolved sugar is evident. Despite deglycosylation, the isoelectric point of the protein is retained and the molecular weight is correspondingly reduced.

Example 3: Preparation of biomatrix 16 ml of collagen solution was placed in a 50 ml-centrifuge tube and placed on ice. Each 600 μl was carefully injected into a well of a microtiter plate with 24 wells (each well has a diameter of 10 mm). The mixture is gelled by incubation at 37 ° C. for 2 minutes.

  Prior to inoculation of cells to be cultured thereon, first carefully aspirate the medium in the wells of the microtiter plate and from the gel.

Example 4: Preparation of fibroblast cultures For functional characterization of the biomatrix produced according to Example 3, fibroblasts were cultured on the matrix. As a comparative batch, a matrix produced by a known method (acetic acid-extraction) was used.

For obtaining fibroblasts, a method known per se was used. Individually, human donor skin (foreskin) was incubated with Dispase-solution and subsequently treated with trypsin solution. The epidermis layer was peeled off from the tissue piece pretreated with the enzyme, and then the dermis was shredded using an anatomical knife and incubated in collagenase type 4 (500 U / ml) at 37 ° C. for 30 to 45 minutes. Subsequently, centrifugation was performed at 1000 rpm for 5 minutes while removing the supernatant. The pellet was resuspended in DMEM + FCS 10% and centrifuged again. After removing the supernatant by aspiration, the pellet was placed in 2 ml DMEM + 10% FCS and transferred into an uncoated cell culture bottle. After 1-2 days of culture, 10-15 ml of DMEM + 10% FCS was additionally supplied and further cultured. The medium was changed every 3 days. Cultivation was performed in a steam saturated incubator under normal cell culture conditions (37 ° C. and 5% CO ₂ -atmosphere).

In a comparative batch, fibroblasts were subcultured and placed in a gel injection solution (consisting of cell culture medium, serum and buffer) at a concentration of 1 × 10 ⁴ cells / cell and obtained by acetic acid extraction. Pipette into collagen solution. Using a pipette / dropper, the mixture was removed, mixed at the same time, and 500 μl of each was pipetted into a 24-well plate. Thereafter, in order to allow complete gelation of the gel, the gel was incubated at 37 ° C. for 5 minutes in an incubator. After complete gelation of this gel, about 1.5 ml of DMEM per well + 10% FCS + 1% gentamicin was added to this gel and cultured under standard culture conditions. After one week, significant shrinkage of the culture was observed in the comparison batch. After a culture time of about 18 days, the experiment in the comparison batch had to be interrupted due to too strong contraction.

On the other hand, in the batch according to the invention, the biomatrix obtained according to the invention is gelled for the time being and subsequently the subcultured cells are added at a concentration of 1 × 10 ⁴ cells / ml in the gel injection solution. As a fibroblast-suspension. Subsequently, the cells were cultured by a method known per se (see comparative experiment). After a culture time of 40 days, it was confirmed that the fibroblasts grew well by the vital staining method. This growth could be clearly observed and visualized after 60 days by microscopic observation and tissue staining (hematoxylin-iocin). From tissue staining, it is also clear that fibroblasts from this biomatrix have synthesized a new matrix (FIG. 1).

Example 5: Production of a multilayer in vitro skin model A skin model consisting of human fibroblasts and primordial keratinocytes was produced.

  Human fibroblasts were obtained as in Example 4. The acquisition of keratinocytes is performed as follows: The epidermis separated from the enzyme-treated tissue pieces was subjected to further trypsin-treatment and subsequently mechanically shredded. Mechanically chopped epidermal particles were placed in special keratinocyte media and / or stopped with trypsin inhibitor. Subsequently, the cells are centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, the pellet is carefully resuspended with 2 ml of keratinocyte medium and transferred into a culture vessel. After about 4 hours, the medium is changed under aseptic conditions. Subsequently, the medium is changed every two days. Cultivation is performed by standard methods known per se under standard culture conditions (see Example 4).

For the production of an in vitro-skin model, a collagen solution obtained according to the invention (see Example 1) and a so-called gel injection solution consisting of cell culture medium, serum and buffer were prepared in advance and placed on ice. First, fibroblasts are subcultured, placed in a gel injection solution at a concentration of about 1 × 10 ⁴ / ml cells, and immediately pipetted into the collagen solution without bubbles. Remove the mixture with a pipette / dropper and mix simultaneously. Pipette 500 μl of each into a 24-well plate (Fa. Nunc) with a special insert. Thereafter, the gel is incubated in an incubator at 37 ° C. for 5 minutes. After complete gelation, add about 1.5 ml DMEM + 10% FCS + 1% gentamicin per well and incubate overnight at 37 ° C. On the next day, the medium is removed by suction. Then 25 ml of fibronectin was pipetted onto the gel at a concentration of 50 μg / mg, and the gel was again placed in an incubator for about 30 minutes.

Then, inoculate keratinocytes. For this purpose, keratinocytes are subcultured from the culture medium, and 1 × 10 ⁵ / ml of cells are pipetted onto the gel in 50 μl / KMr + FCS 5%. Then incubate at 37 ° C. for 20 minutes. Thereafter, this gel is further cultured in submerged culture with KMBr + FCS 5%. In subsequent 5 culture days, the FCS concentration is reduced from 5% to 0%. This further preparation is then cultured for 12-14 days in an aerated environment.

Claims (31)

In a method of isolating collagen or collagen matrix protein from a collagen-containing tissue,
a) isolating collagen-containing fibers from the tissue;
b) incubating the isolated collagen-containing fibers in an aqueous solution containing urea at a final concentration of 5-15 mol / l, wherein the collagen-containing fraction is eluted from the fibers; and c) dissolution Separating the collagen-containing fraction from the fiber and tissue residue to obtain a collagen-containing aqueous solution;
A method of isolating collagen or collagen matrix protein, comprising Further steps:
d) separating urea from the collagen-containing fraction using gradient dialysis;
The method of claim 1, comprising: Further steps:
e) restore collagen;
The method of claim 2, comprising:   4. The method according to claim 3, wherein in step e), reconstitution in the culture medium.   4. The method of claim 3, wherein in step e), reconstitution in PBS.   6. The method according to any one of claims 1 to 5, wherein the collagen-containing tissue is an isolated rat tail tendon.   The method according to any one of claims 1 to 6, wherein the collagen-containing tissue is acellularized porcine small intestine.   8. The process according to claim 1, wherein the final urea concentration in step b) is 7 to 12 mol / l.   9. The process according to claim 1, wherein the urea final concentration in step b) is 9 mol / l.   10. The method according to any one of claims 1 to 9, wherein in step b) the collagen-containing fibers are stirred with the urea solution.   11. A method according to any one of claims 1 to 10, wherein in step b) the collagen-containing fibers are incubated with the urea solution for 12 to 36 hours.   12. The method according to any one of claims 1 to 11, wherein in step c), the dissolved collagen-containing fraction is separated by centrifugation and / or filtration.   13. The process according to claim 1, wherein in step c), the dissolved collagen-containing fraction is separated by fractionation using gel filtration and, optionally, the plurality of fractions are again recombined. Method.   14. The method according to any one of claims 2 to 13, wherein in step d), the mixture is dialyzed against water for 4 to 12 days when cold.   The method according to any one of claims 1 to 14, wherein the obtained aqueous collagen solution has a collagen content of 3 to 8 mg / ml.   The method according to any one of claims 1 to 15, wherein the collagen solution obtained is freeze-dried in another step.   The method according to any one of claims 1 to 16, wherein the collagen content of the obtained aqueous collagen solution is obtained by UV-spectroscopy (UV).   18. The method of claim 17, wherein the characteristic amino acid-signature of histidine at 213 nm is evaluated for concentration measurements in UV-spectrophotographs.   19. Isolated collagen that is or can be produced by the method of any one of claims 1-18.   20. Collagen according to claim 19, showing a triple helix-signature by circular dichroism-spectroscopy (DC).   21. Collagen according to claim 20, wherein the triple helix-signature in the DC-spectrophotograph is characterized by a negative absorption band of 217-227 nm and optionally a weak negative absorption band around 200 nm.   23. Collagen according to any one of claims 19 to 22, showing in UV-spectroscopy (UV) the characteristic amino acid signature of histidine at 213 nm. In a method for producing a biomatrix,
Implementing steps a) to e) of the method according to any one of claims 3 to 18;
f) mixing the resulting collagen solution and cell culture medium in a ratio of 2: 1 to 1: 2 to give a collagen-containing matrix precursor solution; and g) the matrix precursor solution at an elevated temperature. Gelling into a collagen-containing biomatrix;
A process for producing a biomatrix.   A biomatrix that is or can be produced by the method of claim 23.   23. A biomatrix comprising the isolated collagen of any one of claims 19-22.   25. A tissue substitute comprising the biomatrix of claim 23 or 24 and biological tissue cells cultured in and / or on the biomatrix.   25. An organ substitute containing the biomatrix according to claim 23 or 24 and living tissue cells cultured in and / or on the biomatrix.   25. An in vitro test system comprising the biomatrix according to claim 23 or 24 and a living cell cultured in and / or on the biomatrix.   Use of collagen or biomatrix, wherein the collagen according to any one of claims 19 to 22 or the biomatrix according to claim 23 or 24 is used for the production of a tissue substitute.   Use of collagen or biomatrix, wherein the collagen according to any one of claims 19 to 22 or the biomatrix according to claim 23 or 24 is used for the production of an organ substitute.   Use of collagen or biomatrix, wherein the collagen according to any one of claims 19 to 22 or the biomatrix according to claim 23 or 24 is used for the production of an in vitro test system.

Images